PROJECT SUMMARY Cells communicate through physical interaction of cell surface proteins or through autocrine, paracrine, juxtacrine or endocrine mechanisms. However, cell types identified by current large-scale single-cell sequencing and spatial transcriptomics efforts may mask heterogeneity in functional responses between cell types. Our ultimate vision for a product would be a platform to interrogate multiple cell-types in different combinations and under different biochemical conditions to understand heterogenous gene expression responses. In phase I of this SBIR, Scribe Biosciences will adapt its proprietary Printed Droplet Microfluidics (PDM) technology to study the impact of perturbations on single-cell transcriptomics. PDM can precisely deliver reagents and single cells to a microwell array at any time, enabling flexible multi-step workflows while maintaining high experimental throughputs. PDM will be used to encapsulate single-cells, incubate them either with an agonist/antagonist or additional cell(s) of different cell-types, and perform scRNA-seq to understand induced transcriptional changes. A novel lipid-barcoding scheme will be used to link cells encapsulated in the same droplets, enabling cells to be recovered with traceable signatures in bulk and sequenced by established single- cell methods. In Specific Aim 1 we will demonstrate the ability to perturb and incubate single cells in a test system where single Jurkat cells isolated in droplets are treated with anti-CD3 and anti-CD28 antibodies, then assayed for IL2p expression. In Specific Aim 2 we will demonstrate a barcoding method to bioinformatically link cells incubated within the same droplet while allowing them to be sequenced separately. The successful execution of this phase I program will demonstrate the technical feasibility of a high throughput functional genomics platform using our PDM technology and will set the stage for a numerical scale up of the platform to analyze up to 10,000 single cells.